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Eagleson Et Al. Supplement Proteomic and Mitochondrial Eagleson et al. Supplement Proteomic and mitochondrial adaptations to early-life stress differ in juveniles and adults Supplemental Results There was no difference in dam and pup body weight at P2, the start of the ELS period (Supplemental Figures S2A, D; Supplemental Tables S1, S2). Rearing had no effect on body weight gain in dams over the stress period (Supplemental Figure S2B; Supplemental Table S1). In contrast, there was a large effect of rearing (F(1,188) = 49.91, p < .001, 2 = .191), but no effect of sex or sex*rearing interaction, on weight gain in pups over the same period (Supplemental Figure S2E; Supplemental Table S2). ELS pups weighed ~7% less than control pups at P9 (Supplemental Figure S2F; Supplemental Table S2). We also measured offspring weight at P21 and adults, the ages at which our analyses were performed. There was a small effect of rearing (F(1,188) = 8.67, p = .004, 2 = .038), but no effect of sex or sex*rearing interaction, on weight gain between P9 and P21 (Supplemental Figure S2G; Supplemental Table S2); thus ELS pups still weighed ~7% less than control pups at P21 (Supplemental Figure S2H; Supplemental Table S2). By adulthood, there is a large effect of sex (F(1,92) = 305.426, p < .001, 2 = .763), but no effect of rearing or sex*rearing interaction, on offspring weight (Supplemental Figure S2I; Supplemental Table S2), consistent with previous reports (1-4). Eagleson S1 Figure S1. Characterization of the isolated mitochondrial preparation in pilot studies. (A) Western blot analysis confirms enrichment of OXPHOS complexes in isolated mitochondria (Mito) compared to non-nuclear homogenates (Hom) of hippocampus and liver. (B) Mitochondrial integrity was confirmed by measuring citrate synthase activity in the presence and absence of CellLytic M, which lyses the mitochondria. Only lysed mitochondria display CS activity. Eagleson S2 Figure S2. Effects of ELS on body weight gain and total body weight during the stress period and at the age of analysis. For dams, there is no difference in (A) body weight at the start of the ELS period, (B) body weight gain over the ELS period, or (C) body weight at the end of the ELS period. For offspring, there is no difference in body weight at the start of the ELS period (D). Body weight gain over the ELS period is reduced in ELS males and females (E), but returns to control-reared levels after the stress period ends (G). Male and female ELS pups weigh less than control-reared pups at the end of the ELS period (D) and at weaning (E). Weights have normalized by adulthood (F). Eagleson S3 Data are presented as box and whisker plots. The box represents the 25th and 75 percentiles, the whiskers denote the 10th and 90th percentiles, and the dots represent the 5th and 95th percentiles. The line bisecting each box is the median. *Significantly different from control-reared mice of the same sex. Raw data and statistical details are provided in Supplementary Tables S1, S2. Eagleson S4 Supplemental References 1. Rice CJ, Sandman CA, Lenjavi MR, Baram TZ (2008): A novel mouse model for acute and long- lasting consequences of early life stress. Endocrinology. 149:4892-4900. 2. Naninck EF, Hoeijmakers L, Kakava-Georgiadou N, Meesters A, Lazic SE, Lucassen PJ, et al. (2015): Chronic early life stress alters developmental and adult neurogenesis and impairs cognitive function in mice. Hippocampus. 25:309-328. 3. Yam KY, Naninck EF, Abbink MR, la Fleur SE, Schipper L, van den Beukel JC, et al. (2017): Exposure to chronic early-life stress lastingly alters the adipose tissue, the leptin system and changes the vulnerability to western-style diet later in life in mice. Psychoneuroendocrinology. 77:186-195. 4. Kanatsou S, Karst H, Kortesidou D, van den Akker RA, den Blaauwen J, Harris AP, et al. (2017): Overexpression of mineralcorticoid receptors in the mouse forebrain partly alleviates the effects of chronic early life stress on spatial memory, neurogenesis and synaptic function in the dentate gyrus. Front Cell Neurosci. 11:132. Eagleson S5 Table S1. Body weight (g) of control and ELS dams Control (n=48) ELS (n=48) P2 P9 P9-P2 P2 P9 P9-P2 30.4 31.4 1.0 31.0 32.8 1.8 30.8 32.9 2.1 33.1 34.6 1.5 29.9 32.8 2.9 29.7 32.4 2.7 31.6 33.4 1.8 28.7 32.0 3.3 27.1 30.3 3.2 32.3 34.8 2.5 30.9 33.5 2.6 28.4 32.1 3.7 28.9 31.3 2.4 29.2 30.4 1.2 28.8 30.7 1.9 30.4 33.5 3.1 32.4 33.0 0.6 30.0 33.4 3.4 30.6 31.6 1.0 31.8 33.1 1.3 32.0 32.5 0.5 30.7 33.3 2.6 27.6 28.1 0.5 32.9 34.7 1.8 31.5 31.8 0.3 30.4 34.9 4.5 25.1 26.2 1.1 30.2 31.8 1.6 32.5 34.7 2.2 29.2 29.2 0.0 28.9 32.6 3.7 33.2 35.4 2.2 31.1 33.6 2.5 33.6 33.1 -0.5 33.2 35.4 2.2 30.8 32.7 1.9 29.9 30.3 0.4 28.8 30.3 1.5 28.0 30.1 2.1 31.8 35.2 3.4 33.6 34.6 1.0 31.3 33.2 1.9 31.5 33.4 1.9 31.8 31.5 -0.3 30.8 33.7 2.9 33.3 34.9 1.6 30.1 32.9 2.8 29.2 32.1 2.9 32.3 33.9 1.6 31.8 34.2 2.4 30.3 31.3 1.0 29.4 32.1 2.7 30.3 31.5 1.2 30.3 32.9 2.6 33.7 34.6 0.9 29.2 32.1 2.9 30.8 32.2 1.4 28.8 32.8 4.0 31.1 33.2 2.1 31.3 33.3 2.0 32.4 34.3 1.9 31.2 31.3 0.1 29.3 31.9 2.6 30.3 33.5 3.2 29.8 32.1 2.3 31.2 32.2 1.0 29.9 31.9 2.0 30.7 31.7 1.0 31.3 32.9 1.6 29.9 31.9 2.0 29.7 32.5 2.8 31.9 32.8 0.9 30.3 31.0 0.7 30.9 33.1 2.2 34.9 35.7 0.8 32.5 33.0 0.5 32.2 34.8 2.6 28.6 30.8 2.2 30.6 31.7 1.1 28.5 31.0 2.5 30.6 31.3 0.7 30.0 31.7 1.7 35.4 39.8 4.4 28.1 30.2 2.1 28.5 30.7 2.2 29.5 31.6 2.1 30.5 33.3 2.8 29.0 31.3 2.3 28.7 31.1 2.4 28.3 32.5 4.2 30.2 32.6 2.4 30.6 34.2 3.6 30.1 31.2 1.1 29.4 32.7 3.3 29.3 33.6 4.3 30.8 33.6 2.8 Mean 30.61 32.50 1.89 30.50 32.66 2.16 Standard deviation 1.91 2.07 0.99 1.47 1.40 1.14 95% CI 30.07, 31.15 32.01, 33.19 1.61, 2.17 30.08, 30.92 32.26, 33.06 1.84, 2.48 Two-tailed unpaired t -test P2 Mean Difference DF t-Value P-Value C, ELS 0.113 94 0.324 0.747 P9 Mean Difference DF t-Value P-Value C, ELS 0.167 94 -0.462 0.645 P9-P2 Mean Difference DF t-Value P-Value C, ELS 0.279 94 -1.283 0.203 Table S2. Body weight (g) of control and ELS offspring ND, not determined because litter used for P21 studies Control ELS Male Female Male Female P2 P9 P9-P2 P21 P21-P9 Adult P2 P9 P9-P2 P21 P21-P9 Adult P2 P9 P9-P2 P21 P21-P9 Adult P2 P9 P9-P2 P21 P21-P9 Adult 1.73 5.44 3.71 10.20 4.76 ND 1.72 5.40 3.68 9.00 3.60 ND 1.88 4.88 3.00 6.57 1.69 ND 1.98 5.21 3.23 7.80 2.59 ND 1.59 4.83 3.24 9.23 4.40 ND 1.65 5.07 3.42 8.60 3.53 ND 2.07 5.74 3.67 8.73 2.99 ND 1.89 5.52 3.63 8.30 2.78 ND 2.00 5.71 3.71 9.59 3.88 ND 2.07 5.92 3.85 9.78 3.86 ND 1.66 5.33 3.67 8.75 3.42 ND 1.52 4.85 3.33 8.8 3.95 ND 1.54 4.73 3.19 9.38 4.65 28.73 1.56 4.70 3.14 9.25 4.55 23.59 1.49 4.30 2.81 8.80 4.50 31.29 1.45 4.05 2.60 8.70 4.65 23.56 1.57 3.87 2.30 7.83 3.96 31.63 1.60 3.80 2.20 7.97 4.17 24.23 1.61 4.67 3.06 8.87 4.20 28.28 1.51 4.45 2.94 9.15 4.70 19.80 1.56 5.44 3.88 10.00 4.56 28.59 1.43 5.19 3.76 9.52 4.33 20.98 1.38 4.06 2.68 9.85 5.79 28.18 1.37 4.06 2.69 9.48 5.42 23.38 1.70 5.43 3.73 9.65 4.22 ND 1.62 5.13 3.51 9.36 4.23 ND 2.34 5.40 3.06 8.81 3.41 ND 2.31 5.10 2.79 8.89 3.79 ND 1.69 5.35 3.66 9.87 4.52 ND 1.71 5.37 3.66 8.68 3.31 ND 1.57 4.54 2.97 9.76 5.22 ND 1.53 4.60 3.07 9.69 5.09 ND 1.73 5.28 3.55 10.10 4.82 28.82 1.69 5.70 4.01 9.65 3.95 20.83 1.74 4.78 3.04 8.93 4.15 27.39 1.62 4.89 3.27 9.10 4.21 23.53 1.71 5.26 3.55 9.73 4.47 31.31 1.69 5.26 3.57 9.35 4.09 23.51 1.77 5.44 3.67 9.93 4.49 29.01 1.68 5.29 3.61 9.35 4.06 21.23 2.01 6.60 4.59 9.53 2.93 ND 1.82 7.09 5.27 8.05 0.96 ND 1.51 4.44 2.93 8.07 3.63 ND 1.57 4.90 3.33 8.40 3.50 ND 1.69 5.08 3.39 11.63 6.55 ND 1.63 5.15 3.52 10.80 5.65 ND 1.75 5.15 3.40 8.53 3.38 ND 1.79 5.18 3.39 8.40 3.22 ND 2.09 5.37 3.28 9.97 4.60 36.38 1.97 5.53 3.56 10.10 4.57 23.19 1.84 4.37 2.53 7.90 3.53 28.00 2.06 5.70 3.64 8.55 2.85 22.97 1.44 4.35 2.91 10.06 5.71 32.00 1.59 4.60 3.01 9.90 5.30 24.44 1.64 4.39 2.75 8.92 4.53 32.56 1.57 4.98 3.41 8.71 3.73 24.89 1.65 5.58 3.93 8.92 3.34 28.49 1.58 5.53 3.95 9.11 3.58 25.31 1.51 4.31 2.80 8.72 4.41 28.12 1.62 5.09 3.47 9.19 4.10 25.17 1.59 4.53 2.94 9.60 5.07 ND 1.59 4.54 2.95 9.51 4.97 ND 1.54 4.71 3.17 8.64 3.93 ND 1.32 4.40 3.08 8.54 4.14 ND 1.51 4.88 3.37 9.19 4.31 ND 1.50 5.05 3.55 9.29 4.24 ND 1.61 5.54 3.93 9.22 3.68 ND 1.62 5.59 3.97 8.42 2.83 ND 1.73 5.60 3.87 10.03 4.43 27.76 1.67 5.50 3.83 9.50 4.00 22.47 1.79 5.40 3.61 10.03 4.63 27.69 1.73 5.38 3.65 9.63 4.25 23.55 1.70 5.68 3.98 10.00 4.32 32.09 1.73 5.66 3.93 9.47 3.81 24.46 1.76 5.07 3.31 9.73 4.66 27.22 1.72 5.20 3.48 10.23 5.03 23.98 1.66 5.14 3.48 9.33 4.19 ND 1.63 5.24 3.61 9.17 3.93 ND 1.85 5.70 3.85 8.97 3.27 ND 1.99 5.27 3.28 9.40 4.13 ND 1.93 6.47 4.54 9.33 2.86 ND 1.79 5.65 3.86 9.35 3.70 ND 1.76 4.74 2.98 8.40 3.66 ND 1.69 4.56 2.87 8.50 3.94 ND 1.92 5.78 3.86 10.63 4.85 29.01 1.81 5.79 3.98 10.73 4.94 21.12 1.72 4.94 3.22 9.03 4.09 30.31 1.77 5.12 3.35 8.90 3.78 23.45 1.71 5.54 3.83 9.47 3.93 29.76 1.57 5.38 3.81 9.95 4.57 22.45 1.72 5.19 3.47 9.03 3.84 26.93 1.70 5.05 3.35 9.20 4.15 20.81 1.68 5.58 3.90 9.37 3.79 29.95 1.59 5.61 4.02 9.55 3.94 23.82 1.71 4.62 2.91 8.07 3.45 32.80 1.58 4.52 2.94 7.77 3.25 22.68 1.66 5.60 3.94 10.63 5.03 26.43 1.68 5.51 3.83 9.03 3.52 22.95 1.91 4.64 2.73 9.07 4.43 26.67 1.86 4.47 2.61 8.27 3.80 21.64 1.59 5.17 3.58 9.37 4.20 25.88 1.59 5.30 3.71 9.13 3.83 20.41 1.70 4.78 3.08 8.03 3.25 32.39 1.62 4.37 2.75 8.03 3.66 22.33 1.29 4.57 3.28 9.43 4.86 ND 1.20 4.21 3.01 9.46 5.25 ND 1.71 4.73 3.02 9.40 4.67 ND 1.75 4.78 3.03 9.10 4.32 ND 1.61 5.68 4.07 9.67 3.99 ND 1.62 5.66 4.04 10.00 4.34 ND 1.52 5.00 3.48 8.54 3.54 ND 1.44 4.69 3.25 8.60 3.91 ND 1.50 5.09 3.59 9.43 4.34 ND 1.52 5.15 3.63 10.97 5.82 ND 1.66 4.46 2.80 9.20 4.74 ND 1.50 4.41 2.91 9.15 4.74
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